CN109234236A - A kind of preparation method of Chimeric antigen receptor gamma delta T cells - Google Patents
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Abstract
The present invention relates to a kind of preparation methods of Chimeric antigen receptor gamma delta T cells, PBMCs is prepared first, then gamma delta T cells are expanded in vitro, it is subsequently added into CAR virus and polybrene is infected, according to gamma delta T cells purity in the method for the present invention, to determine the preparation CAR- gamma delta T cells time, it does not need additionally to carry out cell purification, simplify operating process, it reduces costs, the method of the present invention is easy to operate, at low cost, and the CAR- gamma delta T cells of preparation have specific tumour lethal effect, and can industrialization production.
Description
Technical field
The invention belongs to field of biotechnology, are related to a kind of system of the Chimeric antigen receptor gamma delta T cells of genetic engineering transformation
Preparation Method.
Background technique
T cell can be divided into according to the difference of cell surface T cell receptor (T cell receptor, TCR) molecular structure
α β T cell and gamma delta T cells.α β T cell is adaptive immunity cell, to the identification of antigen by major histocompatibility complex
The limitation of (Major histocompatibility complex, MHC), and need antigen presenting cell (Antigen
Presenting cell, APC) participation.Since tumour cell often has MHC class molecule loss, α β T cell can not
Tumour cell is effectively removed, it is that leading antitumor cell is immune that this, which is greatly reduced with α β T cell,.Utilize technique for gene engineering
α β T cell is transformed, so that it is expressed Chimeric antigen receptor (Chimeric antigen receptor, CAR), to make
For out not by the α β T cell of MHC and APC limit, i.e. CAR-T cell technology.CAR-T cell technology is in hematological system tumor
In achieve huge breakthrough, using CD19 as the CAR-T cell of target spot be approved by the fda in the United States in 2017 treatment children
With Young adult acute lymphatic leukemia patient.
Although gamma delta T cells and T cell, but be inherent immunity cell, the identification of antigen is not limited by MHC,
Offering for APC is not needed.Although ratio of the gamma delta T cells in periphery blood T lymphocyte is only 5% or so, it joins extensively
It is the important composition portion of human immune system's the first line of defence with the lysises such as allergy, infection, autoimmunity disease and tumour
Point.The clinical research gamma delta T cells infusion that shows to adopt can be obviously improved the life quality of patient.But gamma delta T cells are to tumour
Killing do not have targeting, if it is possible to improve gamma delta T cells and the targeting of tumour cell killed, then will further mention
The clinical value of high gamma delta T cells.Existing research proposes to can be improved gamma delta T in CAR-T technical application to gamma delta T cells
The specific killing of cells against tumor cells.But since ratio is lower in peripheral blood for gamma delta T cells, from peripheral blood
Enough gamma delta T cells are obtained, and the preparation for CAR- gamma delta T cells is not only complicated for operation, at high cost, but also needs to extract and suffer from
The a large amount of peripheral blood of person, brings pain to patient.Although current people can be obtained a large amount of by external evoked amplification technique
Gamma delta T cells, but it have been found that initial stage is expanded in gamma delta T cells, if virus is added in the preparation method according to CAR-T cell,
CAR- gamma delta T cells cannot be not only obtained, but also gamma delta T cells are all difficult to obtain.This shows the preparation method of CAR-T cell simultaneously
It is not suitable for preparing CAR- gamma delta T cells.
Summary of the invention
Present invention aim to address targeting lethal effect of the gamma delta T cells in the prior art to certain tumour cells is weak
The problem of, a kind of simple, quick, low cost preparation Chimeric antigen receptor gamma delta T cells (CAR- gamma delta T) preparation method is provided,
To improve the specificity antineoplastic effect of gamma delta T cells.
Technical solution
A kind of preparation method of Chimeric antigen receptor gamma delta T cells, includes the following steps:
(1) peripheral blood is taken, anticoagulant heparin is separated using lymphocyte separation medium, obtains peripheral blood mononuclear cells
(PBMCs)；
(2) autologous plasma of 5-10v% is added to serum free medium, zoledronic acid, IL-2 and IL-15 is then added,
Gamma delta T cells stimulation culture medium is obtained, has hanged PBMCs using gamma delta T cells stimulation culture medium, and adjust density to 2-4 × 106It is a
Cell/mL, is inoculated in culture bottle, is placed in 37 DEG C, 5%CO2Incubator in cultivate；
(3) when culture is to 3d in the step (2), centrifugation removal stimulation culture medium uses gamma delta T cells amplification culture medium
Cell density is adjusted to 2-4 × 106A cell/mL, later every 2-3d supplement amplification culture medium according to cell density, make cell
Density maintains 2-4 × 106A cell/mL detects gamma delta T cells purity daily, when gamma delta T cells purity is greater than 80%, i.e.,
Gamma delta T cells can be harvested；
The preparation method of the gamma delta T cells amplification culture medium: IL-2 and IL-15 being added into serum free medium, mixes
To obtain the final product；
(4) cell density is adjusted to 3-10 × 10 with gamma delta T cells amplification culture medium by the gamma delta T cells that step (3) obtains6
Then a cell/mL is added CAR virus and polybrene is infected, is placed in 37 DEG C, 5%CO2Incubator in cultivate；
(5) after infection 3-8h in step (4), culture medium is removed to remove virus, and gamma delta T cells amplification training is then added
Feeding base continues to cultivate, and every 2-3d, which adds gamma delta T cells amplification culture medium, makes cell density maintain 2-4 × 106A cell/mL, often
It detects improved gamma delta T cells ratio, and when ratio is greater than 10%, the cell of collection is that Chimeric antigen receptor gamma delta T is thin
Born of the same parents.
In step (2) and (3), the serum free medium is the serum free medium of this field routine, commercially available, than
Such as X-VIVO 15TM, the serum-free T cell culture medium of Optimizer.
Further, in step (2), in the gamma delta T cells stimulation culture medium, zoledronic acid concentration is 1~50 μM, IL-2
Concentration is 200-1000IU/mL, and IL-15 concentration is 5-50ng/mL.
Further, in step (3), added with the autologous plasma of 5-10v% in the serum free medium, be conducive to cell
Amplification, growth.
Further, in step (3), in the gamma delta T cells amplification culture medium, IL-2 concentration is 200-1000IU/mL, IL-
15 concentration are 5-50ng/mL.
Further, in step (3), the method for detecting purity of gamma delta T cells is using flow cytometry.
Further, in step (4), the MOI of the CAR virus is 10-20.The size of MOI and final positive infection efficiency
It is all related with the motility rate of cell.MOI is excessive, and cell infection efficiency may be high, but the number of cell death is more.
Further, in step (4), the final concentration of 2-10 μ g/mL of polybrene.
Further, in step (5), the method for removing culture medium is using centrifugation, and centrifugation rate is 200-300 × g, time
For 5-10min.
The beneficial effects of the present invention are:
The present invention provides a kind of method of the CAR- gamma delta T cells of novel preparation genetic engineering transformation, and in the prior art
Preparation CAR-T cell is compared, and invention removes magnetic beads for purifying process, not only reduces cost, and operation is simpler, only
A small amount of peripheral blood in patients is needed, a large amount of CAR- gamma delta T cells with targeting killing effect can be prepared, be conducive to industrialization
Produce CAR- gamma delta T cells.
Detailed description of the invention
Fig. 1 is the purity result of gamma delta T cells when cultivating 9d in embodiment 1；
Fig. 2 is the streaming result of CAR- gamma delta T cells when cultivating 14d in embodiment 1；
Fig. 3 is the Mortaility results to tumour cell for the CAR- gamma delta T cells that 1 method of embodiment obtains；
The purity result of gamma delta T cells when Fig. 4 is 2 method culture 9d of embodiment；
Fig. 5 is the streaming result of CAR- gamma delta T cells when cultivating 14d in embodiment 2；
Fig. 6 is the Mortaility results to tumour cell for the CAR- gamma delta T cells that 2 method of embodiment obtains.
Specific embodiment
The following describes the present invention in detail with reference to the accompanying drawings and specific embodiments.
In following embodiments, used serum free medium is VIVO-15TMSerum free medium is purchased from Lonza, azoles
Carry out phosphonic acids and is purchased from U.S. day Ni purchased from Jilin Province's Western-style pastry medicine company, IL-2 and IL-15.
Embodiment 1 is by taking CD19 CAR virus as an example
A kind of preparation method of Chimeric antigen receptor gamma delta T cells, includes the following steps:
(1) PBMCs is prepared；
A. the peripheral blood that 50mL patient is extracted using the anticoagulant vacuum blood collection tube of heparin sodium or heparin lithium, in Biohazard Safety Equipment
In, peripheral blood is transferred in disposable 50mL sterile centrifugation tube with disposable 50mL asepsis injector, the centrifugation of 2000 × g room temperature
Blood plasma is transferred in new disposable 50mL sterile centrifugation tube by 10min, and places 30min in 56 DEG C of incubator to go out
Complement living；
B. the physiological saline or PBS of 30-40mL are added in cell precipitation after centrifugation, is uniformly mixed.By what is diluted
Haemocyte is averagely added in 2 separation of lymphocytes pipes containing 15-20mL lymphocyte separation medium；
C. set maximum for the raising speed of centrifuge, reduction of speed degree is set as minimum, then by above-mentioned equipped with lymphocyte point
The centrifuge tube of chaotropic and cell is gently placed in the horizontal hanging basket of centrifuge, and 820 × g room temperature is centrifuged 25min；
D. after being centrifuged, centrifuge tube is gently taken out, and the tunica albuginea among centrifuge tube is gently taken out in Biohazard Safety Equipment
Confluent monolayer cells have hanged cell suspension into new 50mL sterile centrifugation tube, using the physiological saline or PBS of 10 times of volumes, sufficiently mixed
It is even, 8min is centrifuged with 400-500 × g room temperature.After centrifugation, supernatant is discarded, physiological saline is added or PBS has hanged cell,
It is centrifuged 8min with 200-300 × g room temperature, the cell finally obtained is exactly peripheral blood mononuclear cells (PBMCs).
(2) autologous plasma of 5v% is added to serum free medium, zoledronic acid, IL-2 and IL-15 is then added, obtains
Gamma delta T cells stimulation culture medium (gamma delta T cells stimulate in culture medium, and zoledronic acid concentration is 5 μM, and IL-2 concentration is 500IU/mL,
IL-15 concentration is 10ng/mL), PBMCs, adjustment density to 3 × 10 have been hanged using gamma delta T cells stimulation culture medium6A cell/
ML is inoculated in culture bottle, is placed in 37 DEG C, 5%CO2Incubator in cultivate；
(3) when culture is to 3d in the step (2), centrifugation removal stimulation culture medium uses gamma delta T cells amplification culture medium
Cell density is adjusted to 3 × 106A cell/mL, later every 2d supplement amplification culture medium according to cell density, make cell density
Maintain 3 × 106A cell/mL detects gamma delta T cells purity daily, when gamma delta T cells purity is greater than 80%, can harvest
Gamma delta T cells；Fig. 1 is the purity result of gamma delta T cells when cultivating 9d in embodiment 1；This method can be with as can be seen from Figure 1
Obtain the gamma delta T cells of high-purity, ratio 92.91%；
The preparation method of the gamma delta T cells amplification culture medium: into serum free medium be added 5v% autologous plasma,
IL-2 and IL-15, mixes to obtain the final product；In the gamma delta T cells amplification culture medium, IL-2's and 10ng/mL containing 500IU/mL
IL-15。
(4) cell density is adjusted to 5 × 10 with gamma delta T cells amplification culture medium by the gamma delta T cells that step (3) obtains6It is a
Cell/mL is added in 6 orifice plates, the hole 1mL/, and CD19 CAR-T virus is added, and (virus titer is 1 × 109TU/mL, MOI=10)
50 μ L and polybrene (8 μ g/mL of final concentration), are infected, are placed in 37 DEG C, 5%CO2Incubator in cultivate；
(5) after infection 4h in step (4), centrifugation (200-300 × g, 5min) removal culture medium is obtained with removing virus
Improved gamma delta T cells are added new gamma delta T cells amplification culture medium and continue to cultivate, and every 2d adds gamma delta T cells amplification cultivation
Base makes cell density maintain 3 × 106A cell/mL uses Flow cytometry CD19 CAR- gamma delta T cells ratio daily
The streaming result of example, CAR- gamma delta T cells when cultivating 14d is shown in Fig. 2, as seen from Figure 2, can obtain table through this method
Up to the gamma delta T cells of CD19-CAR, ratio 35.88%.
1 vector virus control group of comparative example
Step (1)-(3) and step (5) are same as Example 1, and step is changed in (4): the gamma delta T that step (3) is obtained
Cell density is adjusted to 5 × 10 with gamma delta T cells amplification culture medium by cell6A cell/mL is added in 6 orifice plates, the hole 1mL/, every hole
Control virus is added, and (virus titer is 1 × 109TU/mL, MOI=10) 50 μ L and polybrene (8 μ g/mL of final concentration).Control
Virus is the control virus of empty carrier.CD19 CAR virus is that the CAR gene of CD19 will be resisted to be connected in carrier, is then prepared into slow
Virus.Control virus is exactly to be not connected to the virus of the carrier preparation of CD19 CAR gene.
2 gamma delta T cells group of comparative example
Step (1)-(3) and step (5) are same as Example 1, and step is changed in (4): the gamma delta T that step (3) is obtained
Cell density is adjusted to 5 × 10 with gamma delta T cells amplification culture medium by cell6A cell/mL is added in 6 orifice plates, the hole 1mL/, every hole
50 μ L culture mediums and polybrene (8 μ g/mL of final concentration) are added.
By CD19 CAR- gamma delta T cells made from embodiment 1 carry out efficiency of infection and anti-tumor function detection, and with comparison
The gamma delta T cells of example 1 and comparative example 2 compare.Test method is as follows:
(1) processing of target cell
1) the K562 cell and Raji cell for collecting culture respectively are centrifuged 5min as target cell with 250 × g room temperature, raw
Reason salt water or PBS are washed 1 time；
2) target cell is resuspended with physiological saline or PBS, and its density is adjusted to 1 × 106A cell/mL；
3) 1mL target cell (1 × 10 is taken6A cell/mL), it is added CalceinAM (final concentration of 1 μM), 37 DEG C, 5%CO2
It is protected from light in incubator and is incubated for 25min.
4) target cell being incubated for physiological saline or PBS are washed 2 times, and 250 × g room temperature is centrifuged 5min, with containing 5%FBS
RPMI-1640 culture medium target cell is resuspended, and its density is adjusted to 5 × 104A cell/mL, it is spare.
(2) processing of effector cell
CD19 CAR- gamma delta T cells are taken, 5min is centrifuged with 250 × g room temperature, collects cell, and with containing 5%FBS's
RPMI-1640 culture medium adjusts cell density to 5 × 105A cell/mL (10:1).
(3) bed board
Plank uses sterile 96 orifice plate with cover of round bottom, and target cell and effector cell is added according to following table, respectively sets three multiple holes,
It is averaged.
(4) it is incubated for
It is placed on 37 DEG C, 5%CO2It is protected from light in incubator and is incubated for 4h.
(5) it detects
96 well culture plates are gently taken out, are careful not to hang cell, it, can the centrifugation of 250 × g room temperature if cell hangs
5min.The 100 μ L of gentle aspiration supernatant from every hole is transferred in new flat 96 hole plate, and draw liquid not touch in the process
And board bottom cell.Supernatant fluorescent value (excitation wavelength 485nm, wavelength of transmitted light 528nm) is detected in microplate reader, according to following public affairs
Formula calculates cytotoxicity:
Cytotoxicity (%)=((experimental port-minimum relief hole))/((maximum relief hole-minimum relief hole)) × 100%
Fig. 3 is the active testing result of specificity antineoplastic for the CD19 CAR- gamma delta T cells that 1 method of embodiment obtains,
The CD19 CAR- gamma delta T obtained as seen from Figure 3 is apparently higher than comparative example 1 to the killing activity of the Raji cell of expression CD19
(vector virus control group) and comparative example 2 (gamma delta T cells group), and three groups of gamma delta T cells have no the lethal effect of K562 cell
Notable difference illustrates that the CD19 CAR- gamma delta T cells obtained using this method can significantly improve gamma delta T cells to expression CD19
The specific killing action of tumour cell.
Embodiment 2 is by taking BCMA CAR virus as an example
A kind of preparation method of Chimeric antigen receptor gamma delta T cells, includes the following steps:
Step (1)-(3) and step (5) are same as Example 1, and step is changed in (4): the gamma delta T that step (3) is obtained
Cell density is adjusted to 5 × 10 with gamma delta T cells amplification culture medium by cell6A cell/mL is added in 6 orifice plates, the hole 1mL/, every hole
BCMA CAR-T virus is added, and (virus titer is 2 × 109TU/mL, MOI=10) 25 μ L and polybrene (8 μ g/ of final concentration
mL).The streaming of gamma delta T cells when Fig. 4 is 2 method culture 9d of embodiment is as a result, this method can obtain as can be seen from Figure 4
Obtain the gamma delta T cells of high-purity, ratio 95.86%；The CAR- gamma delta T that Fig. 5 is obtained when being 2 method culture 14d of embodiment is thin
The streaming of born of the same parents is as a result, as seen from Figure 5, can obtain expression BCMA CAR- gamma delta T cells through this method, ratio is
22.91%.
3 vector virus control group of comparative example
Step (1)-(3) and step (5) are same as Example 1, and step is changed in (4): the gamma delta T that step (3) is obtained
Cell density is adjusted to 5 × 10 with gamma delta T cells amplification culture medium by cell6A cell/mL is added in 6 orifice plates, the hole 1mL/, every hole
Control virus is added, and (virus titer is 1 × 109TU/mL, MOI=10) 50 μ L and polybrene (8 μ g/mL of final concentration).
4 gamma delta T cells group of comparative example
Step (1)-(3) and step (5) are same as Example 1, and step is changed in (4): the gamma delta T that step (3) is obtained
Cell density is adjusted to 5 × 10 with gamma delta T cells amplification culture medium by cell6A cell/mL is added in 6 orifice plates, the hole 1mL/, every hole
50 μ L culture mediums and polybrene (8 μ g/mL of final concentration) are added.
By BCMA CAR- gamma delta T cells made from embodiment 2 carry out efficiency of infection and anti-tumor function detection, and with comparison
The gamma delta T cells of example 3 and comparative example 4 compare.Test method is identical as test method in comparative example 2, and test result is shown in Fig. 6.
Fig. 6 is the active testing result of specificity antineoplastic for the CAR- gamma delta T cells that 2 method of embodiment obtains, by Fig. 6
It can be seen that the BCMA CAR- gamma delta T obtained is apparently higher than 3 (carrier of comparative example to the killing activity of the MM1S cell of expression BCMA
Virus control group) and comparative example 4 (gamma delta T cells group), and three groups of gamma delta T cells have no obvious poor to the lethal effect of K562 cell
It is different, it is thin to expression BCMA tumour to illustrate that the BCMA CAR- gamma delta T cells obtained using this method can significantly improve gamma delta T cells
The specific killing action of born of the same parents.
Claims (7)
1. a kind of preparation method of Chimeric antigen receptor gamma delta T cells, which comprises the steps of:
(1) peripheral blood is taken, anticoagulant heparin is separated using lymphocyte separation medium, obtains PBMCs；
(2) autologous plasma of 5-10v% is added to serum free medium, zoledronic acid, IL-2 and IL-15 is then added, obtains
Gamma delta T cells stimulate culture medium, have hanged PBMCs using gamma delta T cells stimulation culture medium, and adjust density to 2-4 × 106It is a thin
Born of the same parents/mL, are inoculated in culture bottle, are placed in 37 DEG C, 5%CO2Incubator in cultivate；
(3) when culture is to 3d in the step (2), centrifugation removal stimulation culture medium will be thin using gamma delta T cells amplification culture medium
Born of the same parents' density is adjusted to 2-4 × 106A cell/mL, later every 2-3d supplement amplification culture medium according to cell density, make cell density
Maintain 2-4 × 106A cell/mL detects gamma delta T cells purity daily, when gamma delta T cells purity is greater than 80%, Ji Keshou
Obtain gamma delta T cells；
The preparation method of the gamma delta T cells amplification culture medium: IL-2 and IL-15 being added into serum free medium, and mixing is
?；
(4) cell density is adjusted to 3-10 × 10 with gamma delta T cells amplification culture medium by the gamma delta T cells that step (3) obtains6It is a thin
Then born of the same parents/mL are added CAR virus and polybrene are infected, are placed in 37 DEG C, 5%CO2Incubator in cultivate；
(5) after infection 3-8h in step (4), culture medium is removed to remove virus, obtains improved gamma delta T cells, γ is added
Delta T cells amplification culture medium continues to cultivate, and every 2-3d, which adds gamma delta T cells amplification culture medium, makes cell density maintain 2-4 × 106
A cell/mL detects improved gamma delta T cells ratio daily, and when ratio is greater than 10%, the cell of collection is inosculating antibody
Original receptor gamma delta T cells.
2. the preparation method of Chimeric antigen receptor gamma delta T cells as described in claim 1, which is characterized in that described in step (2)
Gamma delta T cells stimulate in culture medium, and zoledronic acid concentration is 1~50 μM, and IL-2 concentration is 200-1000IU/mL, IL-15 concentration
For 5-50ng/mL.
3. the preparation method of Chimeric antigen receptor gamma delta T cells as described in claim 1, which is characterized in that described in step (3)
Added with the autologous plasma of 5-10v% in serum free medium.
4. the preparation method of Chimeric antigen receptor gamma delta T cells as described in claim 1, which is characterized in that described in step (3)
In gamma delta T cells amplification culture medium, IL-2 concentration is 200-1000IU/mL, and IL-15 concentration is 5-50ng/mL.
5. the preparation method of Chimeric antigen receptor gamma delta T cells as described in claim 1, which is characterized in that described in step (4)
The MOI of CAR virus is 10-20.
6. the preparation method of Chimeric antigen receptor gamma delta T cells as described in claim 1, which is characterized in that in step (4),
The final concentration of 2-10 μ g/mL of polybrene.
7. the preparation method of Chimeric antigen receptor gamma delta T cells as described in any one of claim 1 to 6, which is characterized in that step
(5) in, the method for removing culture medium is using centrifugation, and centrifugation rate is 200-300 × g, time 5-10min.
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| CN113549158A (en) * | 2021-07-19 | 2021-10-26 | 广州百暨基因科技有限公司 | Fusion protein comprising mutant IL15 and chimeric antigen receptor |
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